CN101598732A - The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA - Google Patents
The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA Download PDFInfo
- Publication number
- CN101598732A CN101598732A CNA2009100550814A CN200910055081A CN101598732A CN 101598732 A CN101598732 A CN 101598732A CN A2009100550814 A CNA2009100550814 A CN A2009100550814A CN 200910055081 A CN200910055081 A CN 200910055081A CN 101598732 A CN101598732 A CN 101598732A
- Authority
- CN
- China
- Prior art keywords
- hole
- elisa
- detection method
- dilution
- discard
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides the ELISA detection method of a kind of red-tide algae toxin okadaic acid OA, it not only can be quick, easy, the content of OA in the working sample delicately, and with low cost.ELISA detection method provided by the invention may further comprise the steps: after (1) envelope antigen is 1: 2000 with carbonate buffer solution with its dilution, and coated elisa plate; (2) seal ELISA Plate after washing plate 3 times; (3) discard confining liquid, wash plate, dilution is the OA standard solution of series concentration and shellfish extract, respectively with isopyknic monoclonal antibody jolting mixing reaction of pressing the anti-OA of dilution in 1: 10000; (4) discard raffinate, wash plate, add the goat anti-mouse igg antibody of HRP mark, diluted by 1: 5000, every hole 100ul, 37 ℃ are incubated 1h down; (5) discard raffinate, wash plate, add colour developing liquid, survey the OD450 value in each hole.
Description
Technical field:
The present invention relates to a kind of ELISA (enzyme linked immunological) detection method, relate in particular to the ELISA detection method of red-tide algae toxin okadaic acid OA.
Background technology:
Diarrhetic shellfish poison (DSP) is a lipoid soluble natural compound that is produced by part algae kind among poisonous red tide algae fin Trentepohlia Dinophysis and the Prorocentrum Prorocentrum, and principal ingredient is okadaic acid (OA) and derivant DTXs thereof.The toxicity symptom that DSP causes comprises diarrhoea, feels sick, vomiting, lower abdominal pain, generally in edible the last several hours, is as short as the 30min outbreak when serious, and does not have the active drug treatment.
Marine site, China South Sea often has the plain poisoning of diarrhetic shellfish poison to take place, and the diarrhetic shellfish poison element has become influence fishery economic development and sanitarian one big obstacle, and therefore, foundation fast, DSP toxin examination accurately and detection method be extremely important.At present, the method for domestic detection OA mainly is mouse method, high performance liquid chromatography (HPLC) and immunoassay technology (ELISA).The mouse method is simple and easy to do, but shortcoming is to distinguish the kind and the structure of toxin, is subjected to interference bigger, the data out of true; The HPLC method can accurately be analyzed the content and the kind of toxin, and detectability can be low to moderate ng/g, but the sample pretreatment process complexity, and the instrument costliness, need special analytical technology personnel; The ELISA method is much simpler than HPLC method sample pre-treatments, and specificity is good, need not purifying and concentrates.In addition, the ELISA method is shorter than the HPLC method running time, once can handle a large amount of samples, has saved great amount of manpower and material resources, has improved work efficiency.And the ELISA method is littler than the investment of HPLC method, is fit to grass-roots unit and uses, and has therefore presented application promise in clinical practice, is subjected to domestic and international researchist's extensive concern.
Summary of the invention:
The objective of the invention is to provide the ELISA detection method of a kind of red-tide algae toxin okadaic acid OA, it not only can be quick, easy, the content of OA in the working sample delicately, and with low cost.
The object of the present invention is achieved like this: the ELISA detection method of a kind of red-tide algae toxin okadaic acid OA is provided, may further comprise the steps:
(1) envelope antigen: after with carbonate buffer solution its dilution being 1: 2000, bag is by 96 hole ELISA Plate, every hole 100ul, and 4 ℃ are spent the night;
(2) discard raffinate, wash plate 3 times with containing 0.05%Tween-20 phosphate buffer PBST, every hole 200ul, after pat dry, with 1% gelatin sealase target, every hole 150ul, 37 ℃ of following insulation 2h;
(3) discard confining liquid, wash plate; With the shellfish extract of dilution for OA standard solution, the blank of series concentration, the shellfish extract that does not add OA and adding OA standard solution, with isopyknic monoclonal antibody jolting mixing of pressing the anti-OA of dilution in 1: 10000, promptly the final dilutability of monoclonal antibody is 1: 20000 respectively; Every hole adds 100ul, and 37 ℃ are incubated 1h down;
(4) discard raffinate, wash plate, add the goat anti-mouse igg antibody of HRP mark, diluted by 1: 5000, every hole 100ul, 37 ℃ are incubated 1h down;
(5) discard raffinate, wash plate, every hole adds 100ul TMB colour developing liquid, reacts 10min under the room temperature, uses 2M H
2SO
4Cessation reaction, every hole 50ul, microplate reader is measured the OD450 value of each micropore.
With inhibiting rate (P/N) is ordinate, is horizontal ordinate drawing standard working curve with log (10*OA concentration); According to the gained Regression Equations, learn IC50, detectability and linear detection range, but and the content of OA in the calculation sample.
Described envelope antigen is that OA and ovalbumin OVA carry out the conjugate OA-OVA that obtains after the chemical modification, and concentration is for being 2.5mg/L.
Described OA standard solution and shellfish extract all dilute with PBST, and the final content of methyl alcohol is 10% in the shellfish extract.
The monoclonal antibody of described anti-OA by reach 1: 1.28 by tiring * 10
6Above immune mouse ascites preparation.
Described inhibiting rate (P/N)=(A
0-A)/A
0* 100%, A is a light absorption value.
Because the present invention is by optimizing a series of conditions, as determining antigen, the suitableeest working concentration of antibody, grope the influence that methanol content reacts ELISA in the OA lysate, selected for best one anti-, two anti-reaction time, and definite OA indirect competitive ELISA typical curve, finally set up the ELISA detection method of a kind of red-tide algae toxin okadaic acid OA, therefore, had the following advantages:
1. envelope antigen adopts dilution in 1: 2000, and antibody can guarantee that immune response is effective after adopting dilution in 1: 20000, makes that again the antigen-antibody consumption is minimum, and cost is saved greatly.
2. methanol content greatly reduces the appearance of false positive phenomenon in this detection method to the influence of ELISA reaction in the research OA lysate.
3. ELISA detection method of the present invention highly sensitive (IC50 is 2.59ng/ml), detectability low (0.45ng/ml), effectively linear detection range wide (0.3125～50ng/ml), the coefficient of variation is 3.03%～8.94%, illustrate that this method repeatability is good, need not when sample detection, to make how parallel sample, both saved reagent cost, saved the consumption of manpower and time again.
Description of drawings:
Fig. 1 is OA concentration-inhibiting rate curve;
Fig. 2 is an OA indirect competitive ELISA typical curve;
Fig. 3 be when not adding OA methanol content to the influence of light absorption value;
Methanol content was to the influence of inhibiting rate when Fig. 4 was adding OA.
Embodiment:
Embodiment 1: raw material is prepared
Used material is OA standard items (available from Taiwan algae bio technology company limiteds) in the present embodiment; Envelope antigen OA-OVA, OA monoclonal antibody, ELIAS secondary antibody HRG-sheep anti-mouse igg (available from Shanghai ancient cooking vessel state Bioisystech Co., Ltd); NBCS, 1% gelatin, ovalbumin (OVA), bovine serum albumin(BSA) (BSA), N-hydroxyl succimide (N-hydroxysuccinimide), N, N dicyclo ethane carbodiimide (N, N-dicyclohexylcarbodiimide, DCC), N, dinethylformamide (N, N-dimethyformamide, DMF), polyglycol (polythylene glycol-4000, PEG), Fu Shi Freund's complete adjuvant, freund 's incomplete adjuvant, tetramethyl benzidine (TMB), dimethyl sulfoxide (DMSO) (DMSO), MES damping fluid etc. be Sigma company product; SP2/0 myeloma cell's (from national academy of agricultural sciences Shanghai veterinary institute); All the other are that homemade analysis is pure.
The preparation of envelope antigen OA-OVA:
Get a certain amount of N-hydroxyl succimide and N, N dicyclo ethane carbodiimide is dissolved in the MES damping fluid, gets 50 μ l then and joins 100 μ l and contain in the DMSO solution of 1mg OA; Other takes by weighing 3mg OVA, with the dissolving of PBS solution; The OA and the micro-pyridine of above-mentioned activation are added in the OVA solution, stirred 6 hours, 4 ℃ are spent the night.Dialysis subsequently, packing ,-20 ℃ of preservations.
The acquisition of OA monoclonal antibody:
The preparation immunogene: get a certain amount of N-hydroxyl succimide and N, N dicyclo ethane carbodiimide is dissolved in the MES damping fluid, gets 50 μ l then and joins 100 μ l and contain in the DMSO solution of 1mg OA; Other takes by weighing 3mg BSA, with the dissolving of PBS solution; The OA and the micro-pyridine of above-mentioned activation are added in the BSA solution, stirred 6 hours, 4 ℃ are spent the night.Dialysis subsequently, packing ,-20 ℃ of preservations.Make immunizing antigen OA-BSA.
Immune mouse: get BALB/c mouse (purchasing) in Shanghai Si Kelai animal used as test Ltd, head exempts to add the injection of complete Freund foot pad by 150 a μ g/ OA-BSA, and booster immunization is once used incomplete Freund's adjuvant instead after three weeks, hypodermic injection, dosage are 200 μ g/; Continue booster immunization every two weeks later on, the full Freund that toos many or too much for use, intramuscular injection, dosage is 200 μ g/.The 4th abdominal cavity booster immunization, dosage are 200 μ g/, without adjuvant.Get spleen behind the 3d, carry out Fusion of Cells.
Fusion of Cells: the aseptic immune mouse spleen cell of getting mixes with 5: 1 volume ratios with the SP2/0 myeloma cell who is in exponential phase, merges according to a conventional method, and fusion agent is PEG.
The screening of positive hybridoma cell: fused cell grows into 1/3 of culture hole area～1/2 o'clock, adopt indirect ELISA method to screen hybridoma step by step, the single clone of mark or 2 clones' (cell clone is separated from each other) cellular incubation hole at first, with 1 μ g/mL OA-OVA coated elisa plate hole, the culture supernatant that adds measured hole respectively, conventional ELISA screens positive hole (P/N＞2.1), then respectively with OA-OVA and BSA bag quilt, carry out the screening once more of positive colony, only the OA-OVA bag is judged to the positive by the hole colour developing, at last the supernatant in the positive hole of primary dcreening operation is made indirect competitive ELISA, judge the influence of 50ng/ml OA to the OD value, detect inhibiting rates through 2～3 times and all reach 100% hole, in time transfer to and cultivate in the single hole and carry out cloning with limiting dilution assay.Through subclone, and carry out enlarged culture, get cell growth state and well promptly under general light microscopic, observing, cell be homogeneous and transparent, structure is not clearly, and the fast clone cell of value-added speed is built the strain liquid nitrogen and preserved.
The production of monoclonal antibody and purifying: get the BLAB/c female mice in 7 ages in week, lumbar injection sterilization norphytane 0.5mL.The inoculation of 7d pneumoretroperitoneum is in the hybridoma of exponential phase, and every mouse injects 1 * 10 respectively
6Individual cell treats that mouse web portion expands, and is dying when motionless, gathers ascites with syringe needle, with ammonium sulfate precipitation method purifying ascites, and continues purifying ascites, the freeze-drying preservation with DEAE cellulose chromatography post.The ascites fluid of collecting is tired with indirect ELISA method mensuration.Tire reach 1: 1.28 * 10
6Above immune mouse ascites promptly can be used as monoclonal antibody.
The acquisition of embodiment 2:OA indirect competitive ELISA typical curve
The OA-OVA conjugate is used as envelope antigen, with carbonate buffer solution its dilution is 1: 2000 after, the bag by 96 hole ELISA Plate, every hole 100ul, 4 ℃ are spent the night; Discard raffinate, wash plate 3 times with phosphate buffer PBST (containing 0.05%Tween-20), every hole 200ul, after pat dry; With 1% gelatin sealase target, every hole 150ul, 37 ℃ are incubated 2h down; Discard confining liquid, wash plate, with PBST with OA standard solution dilution be 200,100,50,25,12.5,6.25,5,2.5,1.25,0.625,0.3125,0.156,0.078ng/ml, 0ng/ml (being blank), respectively with isopyknic monoclonal antibody jolting mixing of pressing dilution in 1: 10000, be that the final dilutability of monoclonal antibody is 1: 20000, every hole adds 100ul, and 37 ℃ are incubated 1h down; Discard raffinate, wash plate, add ELIAS secondary antibody, dilute at 1: 5000 by the concentration that product description is recommended, every hole 100ul, 37 ℃ are incubated 1h down; Discard raffinate, wash plate, every hole adds 100ul colour developing liquid, reacts 10min under the room temperature, uses 2M H
2SO
4Cessation reaction, every hole 50ul, microplate reader is measured the OD450 value.
With inhibiting rate (P/N) is ordinate, is horizontal ordinate with log (10*OA concentration), obtains OA concentration-inhibiting rate curve, as shown in Figure 1.As can be seen from Figure 1, this curve slightly is the serpentine distribution.When the concentration of OA was lower than 0.3125ng/ml, the immune response inhibiting rate was very low, and inhibiting rate almost drops to 0, illustrated that OA can not play immunosuppressive action to reaction under this concentration; When the concentration of OA was higher than 50ng/ml, OA was about the same to immunoreactive inhibition effect, illustrated that this concentration has surpassed the sensing range of this ELISA detection method.Therefore, the valid analysing range of learning OA concentration is drawn OA indirect competitive ELISA typical curve in this scope between 0.3125～50ng/ml, see Fig. 2.
Embodiment 3: the ELISA of red-tide algae toxin okadaic acid OA detects
A, sample process:
The acquisition of shellfish extract:
The shellfish sample is cleaned up, remove the impurity such as silt on surface, drain after shelling, use the high speed dispersion homogenizer biosome homogenate.Take by weighing 2 parts of shellfish meat samples of homogenate, each 5g places clean centrifuge tube respectively, adds weakly acidic methanol solution, and a copy of it is added a certain amount of OA titer, and another part does not add (being negative control).The centrifugal 10min of 3500r/min gets supernatant.Supernatant is added methyl alcohol and normal hexane successively, vortex mixing, static layering.Discard the normal hexane layer then, evaporate to dryness on Rotary Evaporators adds attachment, evaporate to dryness again on the small amount of methanol dissolving wall subsequently.Use 100ul dissolve with methanol residue at last, add 900ul PBST again, mixing.2 parts of extracts carry out ELISA and detect.
B, detection step:
(1) be after the envelope antigen of 2.5mg/L is 1: 2000 with carbonate buffer solution with its dilution with concentration, bag is by 96 hole ELISA Plate, every hole 100ul, and 4 ℃ are spent the night;
(2) discard raffinate, wash plate 3 times with phosphate buffer PBST (containing 0.05%Tween-20), every hole 200ul, after pat dry, with 1% gelatin sealase target, every hole 150ul, 37 ℃ of following insulation 2h;
(3) discard confining liquid, wash plate, with final concentration is 200ng/ml, 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.156ng/ml, 0.078ng/ml, the OA standard solution of 0ng/ml (being blank), 1 part of shellfish extract and 1 part of shellfish extract that adds the OA standard solution, respectively with isopyknic monoclonal antibody jolting mixing of pressing the anti-OA of dilution in 1: 10000, be that the final dilutability of monoclonal antibody is 1: 20000, every hole adds 100ul, and 37 ℃ are incubated 1h down;
(4) discard raffinate, wash plate, add the goat anti-mouse igg antibody of HRP mark, diluted by 1: 5000, every hole 100ul, 37 ℃ are incubated 1h down;
(5) discard raffinate, wash plate, every hole adds 100ul TMB colour developing liquid, reacts 10min under the room temperature, uses 2M H
2SO
4Cessation reaction, every hole 50ul, microplate reader is measured the OD450 value of each micropore.
C, result judge
With inhibiting rate (P/N) is ordinate, is horizontal ordinate drawing standard working curve with log (10*OA concentration).According to the gained Regression Equations, learn IC50, detectability (being that inhibiting rate is 20% o'clock an OA concentration) and linear detection range, but and the content of OA in the calculation sample.
Inhibiting rate (P/N)=(A
0-A)/A
0* 100%, A is the light absorption value of each concentration OA standard solution, wherein A
0The light absorption value in expression blank hole.
The computing method of OA content in the shellfish sample: try to achieve inhibiting rate (P/N)=(A earlier
N-A
P)/A
N* 100%, A wherein
PBe the light absorption value that has added the shellfish extract of OA, A
NIt is the light absorption value that does not add the shellfish extract of OA.Bring this inhibiting rate the regression equation of typical curve into, and then can try to achieve the OA concentration that is contained in the shellfish extract.
The judge actual shellfish method of poisonous (promptly whether containing OA) whether: if it is almost consistent not add the light absorption value in the blank hole in the middle of light absorption value and the typical curve of shellfish extract of OA, then provable this shellfish does not contain toxin OA; If do not add the light absorption value of shellfish extract of OA and the adding in the middle of the typical curve light absorption value of micropore of OA titer equally matched, under the situation of getting rid of other false positive results, can illustrate that then this shellfish contains toxin OA.
Embodiment 4: methanol content is to the influence of the ELISA detection of okadaic acid OA
1. when not adding OA, the influence that methanol content detects ELISA
(1) be after the envelope antigen of 2.5mg/L is 1: 2000 with carbonate buffer solution with its dilution with concentration, bag is by 96 hole ELISA Plate, every hole 100ul, and 4 ℃ are spent the night;
(2) discard raffinate, wash plate 3 times with phosphate buffer PBST (containing 0.05%Tween-20), every hole 200ul, after pat dry, with 1% gelatin sealase target, every hole 150ul, 37 ℃ of following insulation 2h;
(3) discard confining liquid, wash plate, methyl alcohol is mixed with 0,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%11 concentration group, every group establish 3 parallel, with isopyknic monoclonal antibody jolting mixing of pressing the anti-OA of dilution in 1: 10000, promptly the methyl alcohol final concentration is 0,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and the final dilutability of monoclonal antibody is 1: 20000, every hole adds 100ul, and 37 ℃ are incubated 1h down;
(4) discard raffinate, wash plate, add the goat anti-mouse igg antibody of HRP mark, diluted by 1: 5000, every hole 100ul, 37 ℃ are incubated 1h down;
(5) discard raffinate, wash plate, every hole adds 100ul TMB colour developing liquid, reacts 10min under the room temperature, uses 2M H
2SO
4Cessation reaction, every hole 50ul, microplate reader is measured the OD450 value of each micropore, is worth greatly more, illustrates that methyl alcohol is big more to immunoreactive influence.The results are shown in Figure 3.
2. when adding OA, the influence that methanol content detects ELISA
In an anti-reaction link, four groups of solution of methyl alcohol with 40%, 60%, 80% and PBST dilute OA respectively, every group establish 3 parallel, add ELISA Plate again with behind isopyknic monoclonal antibody mixing, promptly the methyl alcohol final concentration is 20%, 30%, 40%.The blank group does not add OA, and all the other steps are the same.Measure OD then respectively
450Value calculates inhibiting rate, and research relatively methanol content is asked the influence that meets competition inhibition ELISA to OA, and selects best OA dilution.The results are shown in Figure 4.
The result:
Methanol content to the influence of ELISA as can be seen from Fig. 3 and Fig. 4, when not adding OA and carrying out immunoassays, light absorption value OD
450Can increase progressively along with the raising of methanol concentration, i.e. how many meetings of methanol content directly influence the measurement result of ELISA reaction blank value, and when the methyl alcohol final concentration was higher than 10%, this trend that increases progressively was more obvious; When adding OA carried out indirect competition immunosupress reaction, the increase of methanol concentration can reduce immunoreactive inhibiting rate.Therefore, when adding the shellfish sample extraction of OA,, all extract, can with methyl alcohol it be dissolved earlier, with buffer solution PBST methanol content is diluted to below 10% again in order to guarantee OA because OA is dissolved in methanol solution; And when ELISA detects, can be directly with PBST dilution OA standard solution.
The ELISA method of detection OA provided by the invention, with low cost, sample pretreatment process is simple, is particularly suitable for field monitoring and great amount of samples rapid screening.The present invention is after optimizing reaction conditions in addition, and linear detection range is 0.3125～50ng/ml, and lowest detectable limit is 0.45ng/ml, IC50 is 2.59ng/ml, and result of study of the present invention proves that this ELISA method working range is wideer, and sensitivity is higher, detectability is also lower, and experimental repeatability is also high.
Claims (4)
1. the ELISA detection method of a red-tide algae toxin okadaic acid OA is characterized in that comprising the steps:
(1) envelope antigen: after with carbonate buffer solution its dilution being 1: 2000, bag is by 96 hole ELISA Plate, every hole 100ul, and 4 ℃ are spent the night;
(2) discard raffinate, wash plate 3 times with containing 0.05%Tween-20 phosphate buffer PBST, every hole 200ul, after pat dry, with 1% gelatin sealase target, every hole 150ul, 37 ℃ of following insulation 2h;
(3) discard confining liquid, wash plate; With the shellfish extract of dilution for OA standard solution, the blank of series concentration, the shellfish extract that does not add OA and adding OA standard solution, with isopyknic monoclonal antibody jolting mixing of pressing the anti-OA of dilution in 1: 10000, promptly the final dilutability of monoclonal antibody is 1: 20000 respectively; Every hole adds 100ul, and 37 ℃ are incubated 1h down;
(4) discard raffinate, wash plate, add the goat anti-mouse igg antibody of HRP mark, diluted by 1: 5000, every hole 100ul, 37 ℃ are incubated 1h down;
(5) discard raffinate, wash plate, every hole adds 100ul TMB colour developing liquid, reacts 10min under the room temperature, uses 2M H
2SO
4Cessation reaction, every hole 50ul, microplate reader is measured the OD450 value of each micropore.
2. detection method as claimed in claim 1 is characterized in that, described envelope antigen is that OA and ovalbumin OVA carry out the conjugate OA-OVA that obtains after the chemical modification, and concentration is 2.5mg/L.
3. detection method as claimed in claim 1 is characterized in that, described OA standard solution and shellfish extract all dilute with PBST, and the final content of methyl alcohol is 10% in the shellfish extract.
4. the ELISA detection method of a red-tide algae toxin okadaic acid OA is characterized in that the monoclonal antibody of described anti-OA, reach 1: 1.28 by tiring * and 10
6Above immune mouse ascites preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100550814A CN101598732A (en) | 2009-07-21 | 2009-07-21 | The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100550814A CN101598732A (en) | 2009-07-21 | 2009-07-21 | The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101598732A true CN101598732A (en) | 2009-12-09 |
Family
ID=41420237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2009100550814A Pending CN101598732A (en) | 2009-07-21 | 2009-07-21 | The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101598732A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104515854A (en) * | 2015-01-06 | 2015-04-15 | 东北农业大学 | Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin |
| CN105785015A (en) * | 2016-04-25 | 2016-07-20 | 浙江大学 | Kit and method for rapidly and highly sensitively detecting OA |
| CN108949848A (en) * | 2018-08-08 | 2018-12-07 | 浙江海洋大学 | A method of it is fermented using marine bacteria and prepares sponge acid |
-
2009
- 2009-07-21 CN CNA2009100550814A patent/CN101598732A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104515854A (en) * | 2015-01-06 | 2015-04-15 | 东北农业大学 | Monoclonal antibody blocking ELISA (enzyme-linked immuno sorbent assay) testing method of clostridium perfringens alpha toxin |
| CN105785015A (en) * | 2016-04-25 | 2016-07-20 | 浙江大学 | Kit and method for rapidly and highly sensitively detecting OA |
| CN108949848A (en) * | 2018-08-08 | 2018-12-07 | 浙江海洋大学 | A method of it is fermented using marine bacteria and prepares sponge acid |
| CN108949848B (en) * | 2018-08-08 | 2021-08-20 | 浙江海洋大学 | Method for preparing sponge acid by using marine bacteria fermentation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106932586A (en) | I type pyrethroid flows delayed immunization time resolved fluorometric quick testing reagent box | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN111057064B (en) | 14-hydroxyl gelsemine hapten and artificial antigen as well as preparation method and application thereof | |
| CN104762267B (en) | Hybridoma AFB1 2A4 and its caused aflatoxin B1 monoclonal antibody | |
| CN109897829B (en) | Hybridoma cell strain secreting monoclonal antibody against citrus yellow dragon germ and application of monoclonal antibody | |
| CN106290889A (en) | The detection method of AFB1 | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN103864928A (en) | Preparation of common antigen monoclonal antibody of Brucella sLPS (lipopolysaccharides) and establishment of c-ELISA (competitive enzyme-linked immuno sorbent assay)method | |
| CN105527441A (en) | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) method for detecting listeria in food on basis of monoclonal antibodies | |
| CN105400742A (en) | Hybridoma cell strain IMI-G12, neonicotinoid pesticide universal monoclonal antibody generated by same and application | |
| CN106872706A (en) | A kind of FPIA method for detecting carbaryl | |
| CN105277708A (en) | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili | |
| CN101609097B (en) | Competitive enzyme-linked immunosorbent assay method of EV71 neutralizing antibody, kit or reagent and prepration method thereof | |
| CN103288872A (en) | Methyl parathion hapten, and preparation method and application thereof | |
| CN101781365A (en) | Artificial antigen of tetraodotoxin and corresponding specific antibody and preparation method and application thereof | |
| CN101368953A (en) | Chemical luminescence ELISA detection reagent kit of ciprofloxacin | |
| CN101598732A (en) | The ELISA detection method of a kind of red-tide algae toxin okadaic acid OA | |
| CN103645322B (en) | A kind of chemical luminescence ELISA detection kit of streptomysin | |
| CN1979169A (en) | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use | |
| CN104388392A (en) | Enrofloxacin monoclonal antibody as well as preparation method and application thereof | |
| CN104560886A (en) | Anti-natamycin monoclonal antibody hybridoma cell strain and application thereof | |
| CN111273015A (en) | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof | |
| CN102942519B (en) | Nitenpyram hapten, artificial antigen and antibody, their preparation methods and application thereof | |
| CN105543177A (en) | Hybridoma cell strain secreting citrus yellow vein clearing virus-resistant monoclonal antibodies and monoclonal antibody application thereof | |
| CN110257342A (en) | One plant of kitasamycin monoclonal antibody hybridoma cell strain SML and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20091209 |